Process for preparing 4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluoro-phenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-((1S,2S)-2-hydroxycyclohexyl) benzamide or a pharmaceutically acceptable salt thereof

ABSTRACT

A process for preparing a compound of formula (I): 
                         
or a pharmaceutically acceptable salt thereof includes reacting a compound of formula (6):
 
                         
(wherein R is H) with 2-aminocyclohexanol or a salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of co-pending U.S.Non-provisional application Ser. No. 15/347,402, filed Nov. 9, 2016,which in turn, is a Continuation-In-Part claiming priority from PCTApplication No. PCT/GB2016/051467, filed May 20, 2016, which in turn,claims priority from European Application No. 15168637.5, filed May 21,2015. Applicants claim the benefits of 35 U.S.C. § 120 as to theNon-provisional U.S. applications and the PCT application, and priorityunder 35 U.S.C. § 119 as to the said European application, and theentire disclosures of all applications are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to compounds useful in the treatment of mycoses,compositions containing it and its use in therapy.

BACKGROUND OF THE INVENTION

The incidence of fungal infections has increased substantially over thepast two decades and invasive forms are leading causes of morbidity andmortality, especially amongst immunocompromised or immunosuppressedpatients. Disseminated candidiasis, pulmonary aspergillosis, andemerging opportunistic fungi are the most common agents producing theseserious mycoses. It is a particular feature of fungi that they are ableto generate an extracellular matrix (ECM) that binds them together andallows them to adhere to their in vitro or in vivo substrates. Thesebiofilms serve to protect them against the hostile environments of thehost immune system and to resist antimicrobial killing (Kaur and Singh,2013).

Pulmonary aspergillosis can be segmented into those patients sufferingwith non-invasive disease versus those with an invasive condition. Afurther sub-division is used to characterise patients who exhibit anallergic component to aspergillosis (known as ABPA; allergicbronchopulmonary aspergillosis) compared with those that do not. Thefactors precipitating pulmonary aspergillosis may be acute, such asexposure to high doses of immuno-suppressive medicines or to intubationin an intensive care unit. Alternatively, they may be chronic, such as aprevious tuberculosis (TB) infection (Denning et al., 2011a). Chroniclung infections with Aspergillus can leave patients with extensive andpermanent lung damage, requiring lifetime treatment with oral azoledrugs (Limper et al., 2011).

A growing body of research suggests that Aspergillus infection may playan important role in clinical asthma (Chishimba et al., 2012;Pasqualotto et al., 2009). Furthermore, recently published work hascorrelated Aspergillus infection with poorer clinical outcomes inpatients with chronic obstructive pulmonary disease (COPD) (Bafadhel etal., 2013). Similarly cross-sectional studies have shown associationsbetween the presence of Aspergillus spp. and Candida spp. in the sputumand worsened lung function (Chotirmall et al., 2010; Agbetile et al.,2012).

Invasive aspergillosis (IA) exhibits high mortality rates inimmunocompromised patients, for example, those undergoing allogenic stemcell transplantation or solid organ transplants (such as lungtransplants). The first case of IA reported in an immunocompromisedpatient occurred in 1953. This event was concurrent with theintroduction of corticosteroids and cytotoxic chemotherapy intotreatment regimens (Rankin, 1953). Invasive aspergillosis is a majorconcern in the treatment of leukaemia and other haematologicalmalignancies given its high incidence and associated mortality. Deathrates usually exceed 50% (Lin et al., 2001) and long term rates canreach 90% in allogeneic hematopoietic stem cell transplantationrecipients, despite the availability of oral triazole medicines(Salmeron et al., 2012). In patients undergoing solid organtransplantation (particularly of the lung), the use of high doses ofsteroids leaves patients vulnerable to infection (Thompson andPatterson, 2008) which is a serious problem. The disease has alsoappeared in less severely immunocompromised patient populations. Theseinclude those suffering with underlying COPD or cirrhosis, patientsreceiving high dose steroids, and individuals fitted with central venouscatheters or supported by mechanical ventilation (Dimopoulos et al.,2012).

Existing anti-fungal medicines are predominantly dosed either orally orsystemically. These commonly exploited routes of delivery are poor fortreating lung airways infections, since drug concentrations achieved atthe site of infection tend to be lower than those in organs. This isespecially so for the liver, which is a site of toxicity: up to 15% ofpatients treated with voriconazole suffer raised transaminase levels(Levin et al., 2007; Lat and Thompson, 2011). Exposure of the liver alsoresults in significant drug interactions arising from the inhibition ofhepatic P450 enzymes (Jeong, et al., 2009; Wexler et al., 2004).

Furthermore, the widespread use of triazoles, both in the clinic and inagriculture has led to a growing and problematic emergence of resistantmycoses in some locations (Denning et al., 2011b; Bowyer and Denning,2014).

It is clearly evident that an urgent medical need exists for novelanti-fungal medicines that deliver improved efficacy and better systemictolerability profiles.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides compound (I)

that is:

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(2-hydroxycyclohexyl)benzamide,or a pharmaceutically acceptable salt thereof (the “compound of theinvention”).

Compound (I) contains two stereogenic centres within the2-aminocyclohexanol radical and is provided in the form of any of itsfour possible stereoisomers, either as a singular stereoisomer or as amixture of stereoisomers in any ratio (including racemic mixtures).

In a preferred aspect, the invention provides Compound (I) in the formof a stereoisomer selected from Compounds (Ia) and (Ib) illustratedbelow, which are the two stereoisomers derived from the enantiomers oftrans-2-aminocyclohexanol, and pharmaceutically acceptable saltsthereof:

In a more preferred aspect the invention provides Compound (Ia) depictedbelow:

that is:

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-((1S,2S)-2-hydroxycyclohexyl)benzamide,or a pharmaceutically acceptable salt thereof.

Suitably, Compound (I) such as Compound (Ia) is provided as a singularstereoisomer. Also of interest is Compound (Ib) as a singularstereoisomer.

Biological data disclosed herein below reveals that Compound (I), andits stereoisomer Compound (Ia) in particular, are potent inhibitors ofAspergillus fumigatus growth in in vitro assays. In immunosuppressedmice, Compound (Ia) demonstrated potent inhibition of Aspergillusfumigatus infections.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 displays the effects of therapeutic treatment with Compound (Ia)on CFU in the lungs of Aspergillus fumigatus infected,immuno-compromised, neutropenic mice.

FIG. 2 shows the effects of therapeutic treatment with Compound (Ia) onserum galactomannan concentrations in Aspergillus fumigatus infected,immuno-compromised, neutropenic mice.

FIG. 3 shows the effects of therapeutic treatment with Compound (Ia) onAspergillus fumigatus DNA content in the lungs of Aspergillus fumigatusinfected, immuno-compromised, neutropenic mice.

DETAILED DESCRIPTION OF THE INVENTION

The compound of the invention may be prepared from commerciallyavailable starting materials by the synthetic methodology depicted below(Scheme 1). Buchwald coupling of a suitably protected piperazinederivative with 4-bromo-2-methylphenol under conditions typicallyemployed for such reactions provides the N-arylated product 1. Asuitable amine protective group (P) for such transformations is aurethane group such as a Boc group (P=CO₂ ^(t)Bu). Those skilled in theart will appreciate that a wide variety of conditions may be used foraffecting transformations of this kind. In particular, palladiumcatalysts and phosphine ligands such as RuPhosG3 and RuPhos areroutinely employed in the presence of a base, for example, cesiumcarbonate or lithium hexamethyldisilazide.

Reaction of the resulting phenol 1 with an appropriate electrophilicderivative of((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methanol(2, X=OH) under basic conditions generates the ether 3. An example ofsuch a compound is the corresponding tosylate (2, X=OTs) which isreadily available, in high enantiomeric purity, from commercial sources.Whilst tosylate is exemplary, X may also be an alternative leavinggroup, such as a halogen, typically chlorine. Selective removal of theamine protective group reveals the mono-substituted piperazine 4. In thecase of a Boc derivative (R=CO₂ ^(t)Bu), the deprotection step istypically undertaken by exposure of the carbamate to strong mineral acidor a strong organic acid, such as TFA, either neat or in the presence ofa solvent, such as DCM.

A second Buchwald coupling of the amine 4 with an alkyl 4-bromobenzoateunder basic conditions and the agency of a catalyst gives rise to theN,N′-bisarylated product 5 in which R′ represents lower alkyl such asC₁₋₅ alkyl e.g. methyl or ethyl. Saponification of the ester 5 isconveniently undertaken by treatment with a base, such an alkali metalhydroxide, in a mixture of water and a suitable aqueous misciblesolvent. Reaction of the acid product 6, with 2-aminocyclohexanol, understandard amide coupling conditions, widely available in the art,provides Compound (I). Each of the four separate stereoisomers ofCompound (I) may be produced by using the corresponding singlestereoisomer of 2-aminocyclohexanol. The corresponding stereoisomers of2-aminocyclohexanol are each commercially available with highstereoisomeric purity.

Protective groups and the means for their removal are described in“Protective Groups in Organic Synthesis”, by Theodora W. Greene andPeter G. M. Wuts, published by John Wiley & Sons Inc; 4th Rev Ed., 2006,ISBN-10: 0471697540. A review of methodologies for the preparation ofamides is covered in: ‘Amide bond formation and peptide coupling’Montalbetti, C. A. G. N. and Falque, V. Tetrahedron, 2005, 61,10827-10852.

Pharmaceutically acceptable salts of compounds of formula (I) include inparticular pharmaceutically acceptable acid addition salts of saidcompounds. The pharmaceutically acceptable acid addition salts ofcompounds of formula (I) are meant to comprise the therapeuticallyactive non-toxic acid addition salts that the compounds of formula (I)are able to form. These pharmaceutically acceptable acid addition saltscan conveniently be obtained by treating the free base form with suchappropriate acids in a suitable solvent or mixture of solvents.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric acids and the like; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, malonic,succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic,ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-aminosalicylic, pamoic acid and the like.

Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The definition of Compound (I) is intended to include all tautomers ofsaid compound.

As the term is used herein, a “singular stereoisomer” of Compound (I) isa stereoisomer provided in a form of both high diastereomeric and highenantiomeric purity, that is substantially free of the other threestereoisomers of Compound (I) that arise by virtue of the presencetherein of the 2-aminocyclohexanol radical. Typically, the singularstereoisomer constitutes at least 98%, 99%, 99.5%, or 99.9% w/w of thecontent of Compound (I) (i.e. the other stereoisomers constitutes lessthan 2%, 1%, 0.5%, or 0.1% w/w of the content of Compound (I).

The definition of Compound (I) is intended to include all solvates ofsaid compound (including solvates of salts of said compound) unless thecontext specifically indicates otherwise. Examples of solvates includehydrates.

The compound of the disclosure includes embodiments wherein one or moreatoms specified are naturally occurring or non-naturally occurringisotopes. In one embodiment the isotope is a stable isotope. Thus thecompounds of the disclosure include, for example deuterium containingcompounds and the like.

The disclosure also extends to all polymorphic forms of the compoundherein defined.

Novel intermediates as described herein such as compounds of formula(3), (4), (5) and (6) and salts thereof, form a further aspect of theinvention. Salts include pharmaceutically acceptable salts (such asthose mentioned above) and non-pharmaceutically acceptable salts. Saltsof acids (e.g. carboxylic acids) include first and second group metalsalts including sodium, potassium, magnesium and calcium salts.

In an embodiment there is provided a pharmaceutical compositioncomprising the compound of the invention optionally in combination withone or more pharmaceutically acceptable diluents or carriers.

Suitably the compound of the invention is administered topically to thelung or nose, particularly, topically to the lung. Thus, in anembodiment there is provided a pharmaceutical composition comprising thecompound of the invention optionally in combination with one or moretopically acceptable diluents or carriers.

Suitable compositions for pulmonary or intranasal administration includepowders, liquid solutions, liquid suspensions, nasal drops comprisingsolutions or suspensions or pressurised or non-pressurised aerosols.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985). The compositions may also conveniently be administered inmultiple unit dosage form.

Topical administration to the nose or lung may be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.Such formulations may be administered by means of a nebuliser e.g. onethat can be hand-held and portable or for home or hospital use (i.e.non-portable). An example device is a RESPIMAT inhaler. The formulationmay comprise excipients such as water, buffers, tonicity adjustingagents, pH adjusting agents, viscosity modifiers, surfactants andco-solvents (such as ethanol). Suspension liquid and aerosolformulations (whether pressurised or unpressurised) will typicallycontain the compound of the invention in finely divided form, forexample with a D₅₀ of 0.5-10 μm e.g. around 1-5 μm. Particle sizedistributions may be represented using D₁₀, D₅₀ and D₉₀ values. The D₅₀median value of particle size distributions is defined as the particlesize in microns that divides the distribution in half. The measurementderived from laser diffraction is more accurately described as a volumedistribution, and consequently the D₅₀ value obtained using thisprocedure is more meaningfully referred to as a Dv₅₀ value (median for avolume distribution). As used herein Dv values refer to particle sizedistributions measured using laser diffraction. Similarly, D₁₀ and D₉₀values, used in the context of laser diffraction, are taken to mean Dv₁₀and Dv₉₀ values and refer to the particle size whereby 10% of thedistribution lies below the D₁₀ value, and 90% of the distribution liesbelow the D₉₀ value, respectively.

According to one specific aspect of the invention there is provided apharmaceutical composition comprising the compound of the invention inparticulate form suspended in an aqueous medium. The aqueous mediumtypically comprises water and one or more excipients selected frombuffers, tonicity adjusting agents, pH adjusting agents, viscositymodifiers and surfactants.

Topical administration to the nose or lung may also be achieved by useof an aerosol formulation. Aerosol formulations typically comprise theactive ingredient suspended or dissolved in a suitable aerosolpropellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon(HFC). Suitable CFC propellants include trichloromonofluoromethane(propellant 11), dichlorotetrafluoromethane (propellant 114), anddichlorodifluoromethane (propellant 12). Suitable HFC propellantsinclude tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).The propellant typically comprises 40%-99.5% e.g. 40%-90% by weight ofthe total inhalation composition. The formulation may compriseexcipients including co-solvents (e.g. ethanol) and surfactants (e.g.lecithin, sorbitan trioleate and the like). Other possible excipientsinclude polyethylene glycol, polyvinylpyrrolidone, glycerine and thelike. Aerosol formulations are packaged in canisters and a suitable doseis delivered by means of a metering valve (e.g. as supplied by Bespak,Valois or 3M or alternatively by Aptar, Coster or Vari).

Topical administration to the lung may also be achieved by use of adry-powder formulation. A dry powder formulation will contain thecompound of the disclosure in finely divided form, typically with an MMDof 1-10 μm or a D₅₀ of 0.5-10 μm e.g. around 1-5 μm. Powders of thecompound of the invention in finely divided form may be prepared by amicronization process or similar size reduction process. Micronizationmay be performed using a jet mill such as those manufactured by HosokawaAlpine. The resultant particle size distribution may be measured usinglaser diffraction (e.g. with a Malvern Mastersizer 2000S instrument).The formulation will typically contain a topically acceptable diluentsuch as lactose, glucose or mannitol (preferably lactose), usually ofcomparatively large particle size e.g. an MMD of 50 μm or more, e.g. 100μm or more or a D₅₀ of 40-150 μm. As used herein, the term “lactose”refers to a lactose-containing component, including α-lactosemonohydrate, β-lactose monohydrate, α-lactose anhydrous, β-lactoseanhydrous and amorphous lactose. Lactose components may be processed bymicronization, sieving, milling, compression, agglomeration or spraydrying. Commercially available forms of lactose in various forms arealso encompassed, for example Lactohale® (inhalation grade lactose; DFEPharma), InhaLac®70 (sieved lactose for dry powder inhaler; Meggle),Pharmatose® (DFE Pharma) and Respitose® (sieved inhalation gradelactose; DFE Pharma) products. In one embodiment, the lactose componentis selected from the group consisting of α-lactose monohydrate,α-lactose anhydrous and amorphous lactose. Preferably, the lactose isα-lactose monohydrate.

Dry powder formulations may also contain other excipients such as sodiumstearate, calcium stearate or magnesium stearate.

A dry powder formulation is typically delivered using a dry powderinhaler (DPI) device. Example dry powder delivery systems includeSPINHALER, DISKHALER, TURBOHALER, DISKUS, SKYEHALER, ACCUHALER andCLICKHALER. Further examples of dry powder delivery systems includeECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER,BREEZHALER/NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN,ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIELdry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN,PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

The compound of the invention is useful in the treatment of mycoses andfor the prevention or treatment of disease associated with mycoses.

In an aspect of the invention there is provided use of the compound ofthe invention in the manufacture of a medicament for the treatment ofmycoses and for the prevention or treatment of disease associated withmycoses.

In another aspect of the invention there is provided a method oftreatment of a subject with a mycosis which comprises administering tosaid subject an effective amount of the compound of the invention.

In another aspect of the invention there is provided a method ofprevention or treatment of disease associated with a mycosis in asubject which comprises administering to said subject an effectiveamount of the compound of the invention.

Mycoses may, in particular, be caused by Aspergillus spp. such asAspergillus fumigatus.

A disease associated with a mycosis is, for example, pulmonaryaspergillosis.

The compound of the invention may be used in a prophylactic setting byadministering the said compound prior to onset of the mycosis.

In another aspect of the invention there is provided a method oftreatment of a subject with an aspergilloma which comprisesadministering to said subject an effective amount of the compound of theinvention. There is also provided a method of preventing recurrence ofan aspergilloma in a subject which comprises administering to saidsubject an effective amount of the compound of the invention.

Subjects include human and animal subjects, especially human subjects.

The compound of the invention is especially useful for the treatment ofmycoses such as Aspergillus fumigatus infection and for the preventionor treatment of disease associated with mycoses such as Aspergillusfumigatus infection in at risk subjects. At risk subjects includepremature infants, children with congenital defects of the lung orheart, immunocompromised subjects (e.g. those suffering from HIVinfection), asthmatics, subjects with cystic fibrosis, elderly subjectsand subjects suffering from a chronic health condition affecting theheart or lung (e.g. congestive heart failure or chronic obstructivepulmonary disease).

The compound of the invention is also useful in the treatment of othermycoses (and the prevention or treatment of disease associatedtherewith) including those caused by Aureobasidium pullulans, Rhizopusoryzae, Cryptococcus neoformans, Chaetomimum globosum, Penicilliumchrysogenum, Fusarium graminerarum, Cladosporium herbarum, Trichophytonrubrum, Candida spp. e.g. Candida albicans, Candida glabrata and Candidakrusei and other Aspergillus spp. e.g. Aspergillus flavus.

The compound of the invention is also expected to be useful in thetreatment of azole resistant mycoses (and the prevention or treatment ofdisease associated therewith) e.g. those caused by azole resistantAspergillus spp. e.g. Aspergillus fumigatus.

The compound of the invention may be administered in combination with asecond or further active ingredient. Second or further activeingredients may, for example, be selected from other anti-fungal agentsincluding azole anti-fungal agents (such as voriconazole, orposaconazole), amphotericin B, an echinocandin (such as caspofungin) andan inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (such aslovastatin, pravastatin or fluvastatin). Other examples of suitableazole anti-fungal agents include itraconazole and isavuconazole.

The second or further active ingredient may, for example, be selectedfrom voriconazole, posaconazole, itraconazole and caspofungin.

Second or further active ingredients include active ingredients suitablefor the treatment or prevention of a mycosis such as Aspergillusfumigatus infection or disease associated with a mycosis such asAspergillus fumigatus infection or conditions co-morbid with a mycosissuch as Aspergillus fumigatus infection.

The compound of the invention may be co-formulated with a second orfurther active ingredient or the second or further active ingredient maybe formulated to be administered separately by the same or a differentroute.

For example, the compound of the invention may be administered topatients already being treated systemically with an anti-fungal, such asvoriconazole or posaconazole or alternatively itraconazole orisavuconazole.

For example, the compound of the invention may be co-formulated with oneor more agents selected from amphotericin B, an echinocandin, such ascaspofungin, and an inhibitor of 3-hydroxy-3-methyl-glutaryl-CoAreductase, such as lovastatin, pravastatin or fluvastatin.

According to an aspect of the invention there is provided a kit of partscomprising (a) a pharmaceutical composition comprising the compound ofthe invention optionally in combination with one or more diluents orcarriers; (b) a pharmaceutical composition comprising a second activeingredient optionally in combination with one or more diluents orcarriers; (c) optionally one or more further pharmaceutical compositionseach comprising a third or further active ingredient optionally incombination with one or more diluents or carriers; and (d) instructionsfor the administration of the pharmaceutical compositions to a subjectin need thereof. The subject in need thereof may suffer from or besusceptible to a mycosis such as Aspergillus fumigatus infection.

The compound of the invention may be administered at a suitableinterval, for example once per day, twice per day, three times per dayor four times per day.

A suitable dose amount for a human of average weight (50-70 kg) isexpected to be around 50 μg to 10 mg/day e.g. 500 μg to 5 mg/dayalthough the precise dose to be administered may be determined by askilled person.

The compound of the invention is expected to have one or more of thefollowing favourable attributes:

potent antifungal activity, particularly activity against Aspergillusspp. such as Aspergillus fumigates, especially following topicaladministration to the lung or nose;

long duration of action in lungs, preferably consistent with once dailydosing;

low systemic exposure following topical administration to the lung ornose; and

acceptable safety profile, especially following topical administrationto the lung or nose.

EXPERIMENTAL SECTION

Abbreviations used herein are defined below (Table 1). Any abbreviationsnot defined are intended to convey their generally accepted meaning.

TABLE 1 Abbreviations ABPA allergic bronchopulmonary aspergillosis aqaqueous ATCC American Type Culture Collection BALF bronchoalveolarlavage fluid BEAS2B bronchial epithelium + adenovirus 12-SV40 hybrid,line 2B Boc tert-butyloxycarbonyl br broad BSA bovine serum albumin CC₅₀50% cell cytotoxicity concentration CFU colony forming unit(s) CLSIClinical and Laboratory Standards Institute COI cut off index concconcentration COPD chronic obstructive pulmonary disease d doublet DCMdichloromethane DMAP 4-dimethylaminopyridine DMEM Dulbecco's ModifiedEagle Medium DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DNAdeoxyribonucleic acid DSS dextran sodium sulphate EBM endothelial cellbasal media ECM extracellular matrix EDCI1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ee enantiomeric excess EGMendothelial cell growth media EUCAST European Committee on AntimicrobialSusceptibility Testing (ES⁺) electrospray ionization, positive modeEtOAc ethyl acetate FAM 6-fluorescein amidite FBS foetal bovine serum GMgalactomannan hr hour(s) HPAEC primary human pulmonary arteryendothelial cells IA invasive aspergillosis i.n. intranasal i.t.intra-tracheal LC-MS/MS liquid chromatography-mass spectrometry Li Heplithium heparin LiHMDS lithium bis(trimethylsilyl)amide (M + H)⁺protonated molecular ion MDA malondialdehyde Me methyl MeCN acetonitrileMeOH methanol MHz megahertz MIC₅₀ 50% of minimum inhibitoryconcentration MIC₇₅ 75% of minimum inhibitory concentration MIC₉₀ 90% ofminimum inhibitory concentration min minute(s) MMD mass median diameterMOI multiplicity of infection MOPS 3-(N-morpholino)propanesulfonic acidm/z: mass-to-charge ratio NCPF National Collection of Pathogenic FungiNMR nuclear magnetic resonance (spectroscopy) NT not tested OD opticaldensity PBS phosphate buffered saline PCR polymerase chain reaction Pprotective group q quartet RT room temperature RP HPLC reverse phasehigh performance liquid chromatography RPMI Roswell Park MemorialInstitute medium RuPhos2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl RuPhosG3(2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium (II)methanesulfonate s singlet satsaturated sc sub-cutaneous SDS sodium dodecyl sulphate t triplet TAMRAtetramethyl-6-carboxyrhodamine TB tuberculosis TE tris-EDTA(ethylenediaminetetraacetic acid) TFA trifluoroactic acid THFtetrahydrofuran TR34/L98H An Aspergillus fumigatus strain containing aleucine-to- histidine substitution at codon 98 and a 34-bp tandem repeatTR46/Y121F/ An Aspergillus fumigatus strain containing a tyrosine-to-phenylalanine T289A substitution at codon 121, a threonine-to-alaninesubstitution at codon 289 and a 46-bp tandem repeat vol volume(s)

General Procedures

All reagents and solvents were obtained either from commercial sourcesor prepared according to the literature citation. Unless otherwisestated all reactions were stirred. Organic solutions were routinelydried over anhydrous magnesium sulfate.

Analytical Methods

Reverse Phase HPLC Methods:

Waters Xselect CSH C18 XP column, 2.5 μm (4.6×30 mm) at 40° C.; flowrate 2.5-4.5 mL min⁻¹ eluted with a H₂O-MeCN gradient containing either0.1% v/v formic acid (Method 1a) or 10 mM NH₄HCO₃ in water (Method 1b)over 4 min employing UV detection at 254 nm. Gradient information:0-3.00 min, ramped from 95% H₂O-5% MeCN to 5% H₂O-95% MeCN; 3.00-3.01min, held at 5% H₂O-95% MeCN, flow rate increased to 4.5 mL min⁻¹; 3.013.50 min, held at 5% H₂O-95% MeCN; 3.50-3.60 min, returned to 95% H₂O-5%MeCN, flow rate reduced to 3.50 mL min⁻¹; 3.60-3.90 min, held at 95%H₂O-5% MeCN; 3.90-4.00 min, held at 95% H₂O-5% MeCN, flow rate reducedto 2.5 mL min⁻¹.

¹H NMR Spectroscopy:

¹H NMR spectra were acquired on a Bruker Advance III spectrometer at 400MHz using residual undeuterated solvent as reference and unlessspecified otherwise were run in DMSO-d₆.

Preparation of Compounds (Ia-d): the Stereoisomers of Compound (I)

The syntheses of optically pure cis and trans 2-amino hexanols have beenpreviously reported (Jacobsen et al., 1997). These materials areavailable in high enentiomeric purity from numerous commercial sourcesand were used as supplied.

Tert-butyl 4-(4-hydroxy-3-methylphenyl)piperazine-1-carboxylate

A flask charged with tert-butylpiperazin-1-carboxylate (19.1 g, 103mmol), 4-bromo-2-methyl phenol (16.0 g, 86.0 mmol), RuPhos (798 mg, 1.71mmol) and RuPhos G3 (1.43 g, 1.71 mmol) was evacuated and backfilledwith nitrogen three times. A solution of LiHMDS (1M in THF, 257 mL, 257mmol) was added via cannula and the reaction mixture was heated at 70°C. for 3 h. After cooling to RT the mixture was quenched by the additionof 1M aq hydrochloric acid (400 mL) at 0° C. and then neutralised withsat aq NaHCO₃ (400 mL). The aq layer was extracted with EtOAc (1×400 mLthen 2×200 mL) and the combined organic extracts were washed with brine(500 mL) and dried. The volatiles were removed in vacuo to give a crudeproduct which was triturated in diethyl ether:hexane (2:1) (750 mL) andcollected by filtration to afford the title compound, Intermediate 1, asa pink solid (20.7 g, 76%); R^(t) 2.07 min (Method 1b); m/z 293 (M+H)⁺(ES⁺); ¹H NMR δ: 1.41 (9H, s), 2.07 (3H, s), 2.86-2.88 (4H, m),3.41-3.43 (4H, m), 6.58-6.66 (2H, m), 6.71 (1H, d) and 8.73 (1H, s).

1-(4-(((3R,5R)-5-((1H-1,2,4-Triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazine

To a solution of intermediate 1 (21.5 g, 66.1 mmol) in DMSO (408 mL) wasadded aq sodium hydroxide (28.3 mL, 3.5 M, 99.0 mmol). The mixture wasstirred at RT for 30 min and was then treated portionwise with((3S,5R)-5-((1H-1,2,4-triazol-1-yl)methyl-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methyl4-methylbenzenesulfonate 2 (ex APIChem,Catalogue Number: AC-8330, 32.7 g, 72.7 mmol). The reaction mixture wasstirred at 30° C. for 18 h, cooled to RT and water (600 mL) was added.The resulting mixture was extracted with EtOAc (3×500 mL) and thecombined organic extracts were washed with sat aq NaHCO₃ (2×500 mL) andwith brine (500 mL) and then dried and evaporated in vacuo to afford abrown oil (approx. 41 g). Analysis of the crude, N-Boc-protected product3 by ¹H NMR indicated that it contained approximately 10 mole % of thealkene elimination product:(R)-1-((2-(2,4-difluorophenyl)-4-methylenetetrahydrofuran-2-yl)methyl)-1H-1,2,4-triazole[A], together with some unreacted starting materials. This crude productwas used in the subsequent step without purification.

The crude urethane 3 was taken up into DCM (260 mL) and treated with TFA(76.0 mL, 991 mmol). After 2 h at RT the reaction mixture wasconcentrated in vacuo to remove most of the volatiles and was thendiluted with DCM (200 mL) and carefully neutralised with sat aq NaHCO₃(500 mL) to pH 7, resulting in the formation of an emulsion. The mixturewas acidified to pH 1 by the addition of 1M hydrochloric acid (250 mL)and DCM (350 mL) was added to form a biphasic mixture (two layers). Theaq phase was separated and retained and the organic phase was extractedwith 1M hydrochloric acid (800 mL). The combined aq layers were basifiedby the addition of 2M aq sodium hydroxide (500 mL) to pH 14 and thenextracted with EtOAc (3×500 mL). The combined organic extracts werewashed with brine (2000 mL) and then dried and evaporated in vacuo toafford the title compound, 4, as a viscous, brown oil (24.6 g, 78%);R^(t) 1.46 min (Method 1a); m/z 470 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.07 (3H,s), 2.15 (1H, dd), 2.36-2.42 (1H, m), 2.52-2.56 (1H, m), 2.79-2.81 (4H,m), 2.87-2.90 (4H, m), 3.66 (1H, dd), 3.73-3.77 (2H, m), 4.04 (1H, t),4.57 (2H, dd), 6.64 (1H, dd), 6.70-6.75 (2H, m), 6.99 (1H, td),7.25-7.33 (2H, m), 7.76 (1H, s) and 8.34 (1H, s).

Methyl4-(4-(4-(((3R,5R)-5-((1H-1,2,4-Triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoate

A flask charged with intermediate 4 (19.1 g, 40.7 mmol),methyl-4-bromobenzoate (10.5 g, 48.8 mmol), RuPhos (0.38 g, 0.81 mmol, 2mol %), RuPhosG3 (0.68 g, 0.81 mmol, 2 mol %) and cesium carbonate (21.2g, 65.1 mmol) was evacuated and refilled with nitrogen three timesbefore DMF (500 mL) was added. The mixture was heated at 90° C. for 18h, cooled to RT and poured into water (300 mL). The resulting solid wascollected by filtration and was washed with water (3×100 mL) and withdiethyl ether (3×75 mL), and then dried in vacuo at 50° C. to give thetitle compound, 5, as a tan solid (22.8 g, 89%); R^(t) 2.56 min (Method1a); m/z 604 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.09 (3H, s), 2.16 (1H, dd),2.36-2.42 (1H, m), 2.53-2.57 (1H, m), 3.11-3.13 (4H, m), 3.43-3.46 (4H,m), 3.67 (1H, dd), 3.74-3.79 (5H, s overlapping over m), 4.04 (1H, dd),4.58 (2H, dd), 6.75 (2H, br s), 6.85 (1H, br d), 7.00 (1H, td), 7.04(2H, d), 7.27-7.34 (2H, m), 7.77 (1H, s), 7.81 (2H, d) and 8.35 (1H, s).

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-Triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoicAcid

To a suspension of intermediate 5 (22.8 g, 37.8 mmol) in DMSO (1000 mL)was added a solution of lithium hydroxide (4.52 g, 188 mmol) in water(100 mL). The mixture was heated at 70° C. for 22 h and was then cooledto RT, poured into water (1000 mL) and acidified to pH 2 by the additionof 1M hydrochloric acid (300 mL). The mixture was cooled in an ice bathfor 2 h and the resulting precipitate was collected by filtration. Thefilter cake was washed with water (2×200 mL) and with diethyl ether(4×200 mL). The crude solid was triturated with THF (150 mL), collectedby filtration and was then washed with diethyl ether (3×100 mL) anddried in vacuo at 50° C. to give the title compound, 6 as an off-whitesolid (19.7 g, 88%); R^(t) 2.28 min (Method 1a); m/z 590 (M+H)⁺ (ES⁺);¹H NMR δ: 2.09 (3H, s), 2.16 (1H, dd), 2.36-2.42 (1H, m), 2.52-2.58 (1H,m), 3.11-3.14 (4H, m), 3.41-3.44 (4H, m), 3.67 (1H, dd), 3.74-3.79 (2H,m), 4.04 (1H, dd), 4.58 (2H, dd), 6.75 (2H, br s), 6.85 (1H, br d),6.97-7.03 (3H, m), 7.26-7.34 (2H, m), 7.77-7.80 (3H, m), 8.34 (1H, s)and 12.32 (1H, s).

Compound (1a):4-(4-(4-(((3R,5R)-5-((1H-1,2,4-Triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-((1S,2S)-2-hydroxycyclohexyl)benzamide

To a mixture of intermediate 6 (100 mg, 0.17 mmol), EDCI (65 mg, 0.34mmol) and DMAP (2.07 mg, 0.017 mmol) in pyridine (1.0 mL) was added(1S,2S)-2-aminocyclohexanol hydrochloride (51.4 mg, 0.34 mmol). Thereaction mixture was stirred at RT for 16 h and was then diluted withDCM (8.0 mL) and washed with 1M hydrochloric acid (2.0 mL). The mixturewas passed through a phase separator and the organics were evaporated invacuo. The crude product so obtained was purified by flash columnchromatography (SiO₂, 12 g, 0-5% MeOH in EtOAc, gradient elution) toafford the title compound, (Ia) as a white solid (75 mg, 64%).

Preparation of Compounds (1b-1d)

The remaining compound examples of the invention were prepared in asimilar manner by coupling the benzoic acid intermediate 6 with theappropriate single stereoisomer of 2-amino cyclohexanol. These compoundsare readily available from commercial sources. The materials obtainedfrom Sigma Aldrich were supplied as the hydrochloride salts for whichthe following enantiopurities were specified: (1S,2S) trans isomer, >98%ee; (1R, 2R) trans isomer, >98% ee; (1R,2S) cis isomer>97% ee; (1S,2R)cis isomer, >97% ee.

The LCMS and ¹H NMR spectral data for compound examples (1a-1d) arepresented below (Table 2).

TABLE 2 Analytical and Spectral Data for the Compounds of the Invention

R Example No., Name, LCMS and NMR Data

1a:4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-((1S,2S)-2-hydroxycyclohexyl) benzamide. R¹ 2.15 min (Method 1a); m/z 687 (M + H)⁺(ES⁺); ¹H NMR δ: 1.15-1.28 (4H, m), 1.61-1.65 (2H, m), 1.82-1.92 (2H,m), 2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m),3.12-3.15 (4H, m), 3.36-3.43 (5H, m), 3.55-3.62 (1H, m), 3.68 (1H, dd),3.74-3.79 (2H, m), 4.05 (1H, dd), 4.53-4.62 (3H, m), 6.75 (2H, br s),6.85 (1H, br s), 6.97-7.02 (3H, m), 7.25-7.34 (2H, m), 7.76-7.82 (4H,m), 8.34 (1H, s).

1b:4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-((1R,2R)-2-hydroxycyclohexyl)benzamide. R¹ 2.14 min (Method 1a); m/z 687 (M + H)⁺(ES⁺); ¹H NMR δ: 1.15-1.28 (4H, m), 1.61-1.65 (2H, m), 1.82-1.92 (2H,m), 2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m),3.12-3.15 (4H, m), 3.36-3.43 (5H, m), 3.55-3.62 (1H, m), 3.68 (1H, dd),3.74-3.79 (2H, m), 4.05 (1H, dd), 4.53-4.62 (3H, m), 6.75 (2H, br s),6.85 (1H, br s), 6.97-7.02 (3H, m), 7.25-7.34 (2H, m), 7.76-7.82 (4H,m), 8.34 (1H, s).

1c:4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyhpiperazin-1-yl)-N-((1S,2R)-2-hydroxycyclohexyl)benzamide. R¹ 2.46 min (Method 1b); m/z 687 (M + H)⁺(ES⁺); ¹H NMR δ: 1.23-1.34 (2H, m), 1.41-1.74 (6H, m), 2.10 (3H, s),2.16 (1H, dd), 2.37-2.42 (1H, m), 2.52-2.58 (1H, m), 3.12-3.14 (4H, m),3.36-3.38 (4H, m), 3.68 (1H, dd), 3.74-3.82 (4H, m), 4.05 (1H, dd), 4.58(2H, dd), 4.68 (1H, d), 6.75 (2H, br s), 6.85 (1H, br s), 6.98-7.02 (3H,m), 7.26-7.34 (2H, m), 7.51 (1H, d), 7.75-7.77 (3H, m), 8.34 (1H, s).

1d:4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyhpiperazin-1-yl)-N-((1R,2S)-2-hydroxycyclohexyl)benzamide. R¹ 2.46 min (Method 1b); m/z 687 (M + H)⁺(ES⁺); ¹H NMR δ: 1.23-1.34 (2H, m), 1.41-1.74 (6H, m), 2.10 (3H, s),2.16 (1H, dd), 2.37-2.42 (1H, m), 2.52-2.58 (1H, m), 3.12-3.14 (4H, m),3.36-3.38 (4H, m), 3.68 (1H, dd), 3.74-3.82 (4H, m), 4.05 (1H, dd), 4.58(2H, dd), 4.68 (1H, d), 6.75 (2H, br s), 6.85 (1H, br s), 6.97-7.02 (3H,m), 7.25-7.34 (2H, m), 7.50 (1H, d), 7.75-7.77 (3H, m), 8.34 (1H, s).

Biological Testing: Experimental Methods

Assessment of Planktonic Fungus Growth: Broth Microdilution Assay

This assay was conducted using a modified method published by EUCAST(Rodriguez-Tudela, et al., 2008). Spores of Aspergillus fumigatus(NCPF2010, NCPF7010 [methionine 220 mutation], NCPF7099 [Glycine G54mutation]) from Public Health England, Wiltshire; TR34/L98H mutants fromSt Louis Hospital, Paris, France; TR46/Y121F/T289A mutants fromUniversity of Delhi, Delhi, India) were cultured in Sabouraud dextroseagar for 3 days. A stock spore suspension was prepared from a Sabourauddextrose agar culture by washing with PBS-tween (10 mL; phosphatebuffered saline containing 0.05% Tween-20, 100 U/mL penicillin and 100U/mL streptomycin). The spore count was assessed using a Neubauerhaemocytometer and then adjusted to 10⁶ spores/mL with PBS. A workingsuspension of spores (2×10⁵ spores/mL) was prepared in filtersterilised, BSA MOPS RPMI-1640 (50 mL; RPMI-1640 containing 2 mML-glutamine, 0.5% BSA, 2% glucose, 0.165 M MOPS, buffered to pH 7 withNaOH).

For the assay, BSA MOPS RPMI-1640 (50 μL/well) was added throughout the384-well plate (Catalogue number 353962, BD Falcon, Oxford, UK) first.Test compounds (0.5 μL DMSO solution) were then added in quadruplicateusing an Integra VIAFLO 96 (Integra, Zizers, Switzerland), and mixedwell using a plate mixer. Subsequently 50 μL of the working sporesuspension prepared above was added to all wells except non-sporecontrol wells. For non-spore control wells, BSA MOPS RPMI-1640 solution(50 μL/well) was added instead. The plate was covered with a plasticlid, and incubated (35° C. with ambient air) for 48 hr. The OD of eachwell at 530 nm was determined using a multi-scanner (Clariostar: BMG,Buckinghamshire, UK). The percentage inhibition for each well wascalculated and the MIC₅₀, MIC₇₅ and MIC₉₀ values were calculated fromthe concentration-response curve generated for each test compound.

Fungus panel screening was conducted by Eurofins Panlabs Inc. The MICand MIC₅₀ values of the test articles were determined following theguidelines of the CLSI: broth microdilution methods for yeast (CLSIM27-A2), (CLSI, 2002) and for filamentous fungi (CLSI M38-A), (CLSI,2008).

Aspergillus fumigatus Infection of Bronchial Epithelial Cells

BEAS2B cells were seeded in 96-well plates (100 μL; 3×10⁴ cells/well;Catalogue No 3596, Sigma Aldrich, Dorset, UK) in 10% FBS RPMI-1640 andwere then incubated (37° C., 5% CO₂) for one day before experimentation.Test compounds (0.5 μL DMSO solution) or vehicle (DMSO) were added toeach well to give a final DMSO concentration of 0.5%. BEAS2B cells wereincubated with test compounds for 1 hr (35° C., 5% CO₂) before infectionwith Aspergillus fumigatus (20 uL; Public Health England) conidiasuspension (0.5×10⁵/mL in 10% FBS RPMI-1640). The plate was incubatedfor 24 hr (35° C., 5% CO₂). Supernatant (50 uL) was collected andtransferred to a PCR plate (Catalogue No L1402-9700, Starlab, MiltonKeynes, UK), which was frozen (−20° C.) until use. After thawing,supernatant (5 μL) was diluted 1:20 by adding R7-PBS solution (95 μL;1:4 R7 to PBS; Bio-Rad Laboratories, Redmond, Wash., USA). Galactomannanlevels in these samples (50 μL) were measured using Platelia GM-EIA kits(Bio-Rad Laboratories, Redmond, Wash., USA). The percentage inhibitionfor each well was calculated and the IC₅₀ value was calculated from theconcentration-response curve generated for each test compound.

Aspergillus fumigatus Infection of Human Alveoli Bilayers

In vitro models of human alveoli, consisting of a bilayer of humanalveolar epithelial cells and endothelial cells, were prepared aspreviously described (Hope et al., 2007). This system allowsadministration of a test compound to the upper (“air” space) and/orlower (“systemic” space) compartments. This flexibility has beenexploited to explore the effects of combination treatment by dosingCompound (I) to the upper chamber and posaconazole or other anti-fungalagents to the lower chamber. Primary human pulmonary artery endothelialcells (HPAEC) were harvested and diluted to 10⁶ cells/mL in EGM-2 media(Lonza, Basel, Switzerland). Transwells were inverted and the cellsuspension (100 μL/well) applied to the base of each transwell. Theinverted transwells were incubated at RT within a flow hood for 2 hrafter which they were turned upright. EGM-2 media was added to the lower(700 μL/well) and upper (100 μL/well) compartments and the transwellswere incubated for 48 hr (37° C., 5% CO₂). The EGM-2 media in the lowercompartment was then replaced with fresh EGM-2 media. A549 cells wereharvested and diluted to 5×10⁵ cells/mL in 10% EBM, then added to theupper compartment (100 μL/well) of all transwells and the platesincubated for 72 hr (37° C., 5% CO₂). Conidia of itraconazole sensitiveAspergillus fumigatus s (NCPF2010) and itraconazole resistant(TR34-L98H) strains were cultured separately in Sabouraud dextrose agarfor 3 days. A stock conidia suspension of either strain was preparedfrom a Sabouraud dextrose agar culture by washing with PBS-tween (10 mL;PBS containing 0.05% Tween-20, 100 U/mL Penicillin and 100 U/mLStreptomycin). The conidia count was assessed using a Neubauerhaemocytometer and adjusted to 10⁶ conidia/mL with PBS. A working stockof conidia was prepared in EBM (10⁵ conidia/mL) immediately prior touse.

Test and reference compounds (or neat DMSO as the vehicle) were added tothe appropriate wells of 24-well plates (3 μL/well containing 600 μL of2% FBS EBM) for lower compartment treatment and to 96-well plates (1μL/well containing 200 μL of EBM [no FBS]) for the treatment of theupper compartment, to provide a final DMSO concentration of 0.5%. Themedia in the upper compartment was aspirated and that containing theappropriate test and reference compounds, or vehicle, were added (100μL/well). Transwells were then transferred into the 24-well platecontaining the test and reference compounds or DMSO vehicle. Afterincubation for 1 hr (35° C., 5% CO₂) the conidia suspension (10 μL/well)was added to the upper compartment of each transwell. Plates were thenincubated for 24 hr (35° C., 5% CO₂). Supernatants from each compartment(5 μL/compartment) were collected and stored (−20° C.). Media wasreplaced daily after collection of the supernatants and all wells weretreated with test and reference compounds or with DMSO, as describedabove, for 3 days. Samples continued to be collected until fungal growthwas visible by eye in all transwells. The levels of GM in thesupernatant in lower compartment were then measured by ELISA (BioRad,Calif., USA) as an index of Aspergillus fumigatus invasion.

Cell Viability: Resazurin Assay

BEAS2B cells were seeded in 384-well plates (100 μL; 3000/well/; BDFalcon, Catalogue No 353962) in RPMI-LHC8 (RPMI-1640 and LHC8 mediacombined in equal proportions) one day before experimentation. Forcell-free control wells, RPMI-LHC8 (100 μL) was added. Test compounds(0.5 μL of a DMSO solution) were added to give a final DMSOconcentration of 0.5% using an Integra VIAFLO 96 (Integra, Zizers,Switzerland). BEAS2B cells were incubated with each test compound for 1day (37° C./5% CO₂ in RPMI-LHC8). After addition of resazurin stocksolution (5 μL, 0.04%) the plates were incubated for a further 4 hr (37°C./5% CO₂). The fluorescence of each well at 545 nm (excitation) and 590nm (emission) was determined using a multi-scanner (Clariostar: BMGLabtech). The percentage loss of cell viability was calculated for eachwell relative to vehicle (0.5% DMSO) treatment. Where appropriate, aCC₅₀ value was calculated from the concentration-response curvegenerated from the concentration-response curve for each test compound.

In Vivo Anti-fungal Activity

Aspergillus fumigatus (ATCC 13073 [strain: NIH 5233], American TypeCulture Collection, Manassas, Va., USA) was grown on Malt agar (NissuiPharmaceutical, Tokyo, Japan) plates for 6-7 days at RT (24±1° C.).Spores were aseptically dislodged from the agar plates and suspended insterile distilled water with 0.05% Tween 80 and 0.1% agar. On the day ofinfection, spore counts were assessed by haemocytometer and the inoculumwas adjusted to obtain a concentration of 1.67×10⁸ spores mL⁻¹ ofphysiological saline. To induce immunosuppression and neutropenia, A/Jmice (males, 5 weeks old) were dosed with hydrocortisone (Sigma H4881;125 mg/kg, sc,) on days 3, 2 and 1 before infection, and withcyclophosphamide (Sigma C0768; 250 mg/kg, i.p.) 2 days before infection.On day 0, animals were infected with the spore suspension (30 μLintra-nasally).

Test substances were administered intra-nasally (35 μL of a suspension(0.0032-10.0 mg/mL of drug substance in physiological saline) oncedaily, on days 1, 2 and 3 only (thereby representing a therapeutictreatment regimen). For extended prophylactic treatment, test compounds(35 μL of a suspension of 0.0032 or 0.016 mg/mL in physiological saline)were administered intra-nasally once daily for seven days. One group wasdosed 30 min before infection on day 0 but not subsequently, while asecond group was further dosed on days 1, 2 and 3 after infection. Theeffects of these treatment paradigms were compared with those obtainedwhen treatment was restricted in other groups to either one day or 30min before inoculation and then on days 1, 2 and 3 post infection inboth cases. In a final group, dosing was limited still further withanimals dosed twice, one day and 30 min before infection only.

Animal body weights were monitored daily and at 6 hr after the last doseof drug was administered on day 3, the animals were anesthetised, thetracheas cannulated and BALF blood and lung tissue were collected. Thelevels of IL-6 and TNFα in serum were determined using Quantikine® mouseIL-6 or TNF-α ELISA kit (R&D systems, Inc., Minneapolis, Minn., USA)respectively. Aspergillus GM in serum was determination using PlateliaGM-EIA kits (Bio-Rad Laboratories, Redmond, Wash., USA). Cut-off index(COI) was calculated by the formula: Cut-off index=OD in sample/OD incut-off control provided in kit. For tissue fungal load assays, 100 mgof lung tissue was removed aseptically and homogenized in 0.2 mL of 0.1%agar in sterile distilled water. Serially diluted lung homogenates wereplated on Malt agar plates (50 μL/plate), and incubated at 24±1° C. for72 to 96 hr. The colonies of A. fumigatus on each plate were counted andthe fungal titre presented herein as CFUs per gram of lung tissue.

For determination of Aspergillus fumigatus DNA content, DNA wasextracted from either infected lungs or Aspergillus fumigatus with theIsoplant (Nippon Gene) according to the manufacture's instruction. Thetissues cut to <3 mm in any length were mixed with solution I(extraction buffer: 300 μL). Solution II (lysis buffer; benzyl chloride:150 μL) was then added to the mixtures followed by mixing with a vortexmixer for 5 seconds. After incubation at 50° C. for 15 min, solution III(sodium acetate, pH5.2: 150 μL) was added the mixtures agitatedvigorously for 1-3 seconds and then incubated on ice for 15 min. Afterthe incubation, the mixtures were centrifuged at 12,000 g for 15 min at4° C. DNA in the upper aq phases of the supernatants was precipitatedwith 100% ethanol (×2.5 vol), washed with 70% ethanol and dissolved in5-10 μL of TE buffer.

DNA amplification was performed with Premix Ex Taq™ (Takara Bio) in the96-well optical reaction plate. Aspergillus fumigatus 18S rRNA genefragments were amplified with the primer pair; 5′-GGCCCTTAAATAGCCCGGT-3′(SEQ ID No. 1) and 5′-TGAGCCGATAGTCCCCCTAA-3′ (SEQ ID No. 2), andhybridization probe; 5′-FAM-AGCCAGCGGCCCGCAAATG-TAMRA-3′ (SEQ ID No. 3).Real time PCR was performed in a reaction solution (25 μL) contained 50ng of mouse lung DNA with 200 nM of probe) under the followingconditions: initial incubation for 2 min at 50° C., initial denaturationfor 10 min at 95° C., followed by 55 cycles of 15 seconds at 95° C. and1 min at 65° C. The amounts of Aspergillus fumigatus DNA in 50 ng ofmice lung DNA was evaluated from the standard curve with 0.05-50,000 pgof DNA from Aspergillus fumigatus.

Summary of Screening Results

Compound (I) exhibits potent inhibitory activity against planktonicfungal growth as evaluated in a broth microdilution assay (Table 3).

TABLE 3 The Effects of Treatment with Voriconazole, Posaconazole,Amphotericin B and Compounds (Ia-d)) on planktonic fungal growth ofisolates of Aspergillus fumigatus. Treatment MIC₇₅ Values (nM) (Testagainst the indicted Aspergillus fumigatus isolates¹ Compound) NCPF2010L98H TR46 NCPF7099 NCPF7100 Voriconazole 511 2585 >2860 113 543Posaconazole 10.9 98.3 414 167 59.7 Amphotericin B 407 195 187 248 523Compound (Ia) 2.81 12.7 93 10.0 12.7 Compound (Ib) 8.02 303 334 86.287.3 Compound (Ic) 2.27 54.9 164 11.5 10.9 Compound (Id) 8.51 70.0 31611.7 25.2 Table Footnotes: ¹Broth microdilution assay, n = 2-3.

In these assays, Compound (Ia) in particular showed significantlygreater potency versus both the posaconazole-resistant strains(NCPF7099, NCPF7100, TR34/L98H and TR46/Y121F/T289A), as well as theposaconazole-sensitive strain (NCPF2010), than did posaconazole,voriconazole and Amphotericin B.

Compounds (Ia and Ic) also demonstrate potent inhibitory activityagainst fungal infection of bronchial epithelial cells (Table 4). Inthis assay system Compounds (Ia and Ic) showed significantly greaterpotency than voriconazole, and greater potency than posaconazole.Incubation with Compounds (Ia, Ib, Ic and Id) had no or little effect onthe viability of BEAS2B bronchial epithelial cells at concentrationsindicated below.

TABLE 4 The Effects of Treatment with Voriconazole, Posaconazole,Amphotericin B and Compounds (Ia-d) on Aspergillus fumigatus (NCPF2010)planktonic fungal growth, on fungal infection of BEAS2B bronchialepithelial cells and on BEAS2B cell viability. MIC₅₀/CC₅₀ Values fortreatment indicated (nM) Infection of BEAS2B Treatment BEAS2B cells¹Cell Viability² (Test Compound) MIC₅₀ CC₅₀ Voriconazole 154 >28600Posaconazole 11.6 >14300 Amphotericin B nt 2343 Compound (Ia)6.35 >14600 Compound (Ib) nt >14600 Compound (Ic) 1.86 >14600 Compound(Id) nt >14600 Table Footnotes: ¹Bronchial epithelial cells; n = 1-3; ²n= 3; nt: not tested.

The effects of Compound (I) on the growth of a wide range of fungalpathogens was evaluated using the CLSI broth microdilution methods.Compound (Ia) was found to be a potent inhibitor of the growth ofAureobasidium pullulans, Rhizopus oryzae, Cryptococcus neoformans,Chaetomimum globosum, Penicillium chrysogenum, Fusarium graminerarum(Gibberella zeae), Cladosporium herbarum and Trichophyton rubrum as wellas some Candida spp. (notably Candida albicans, Candida glabrata andCandida krusei) and some Aspergillus spp. (notably Aspergillus flavus)(Table 5).

TABLE 5 The inhibitory effects of Compound (Ia) on the growth of a rangeof fungal species. Compound (Ia) Voriconazole Posaconazole Fungal MIC₅₀MIC₁₀₀ MIC₅₀ MIC₁₀₀ MIC₅₀ MIC₁₀₀ Agent Strain (μg/mL) (μg/mL) (μg/mL)Aspergillus ATCC204304 0.13 0.13 1.0 2.0 0.063 0.13 flavus AureobasidiumATCC9348 0.25 1.0 >8.0 >8.0 0.25 1.0 pullulans Candida 20240.0470.016 >2.0 0.031 >8.0 0.031 >8.0 albicans ATCC10231 0.25 >2.0 0.25 >8.00.13 >8.0 20183.073 0.125 >2.0 4.0 >8.0 0.25 >8.0 Candida ATCC365830.25 >2.0 0.25 >8.0 0.5 >8.0 glabrata 20197.1 0.0078 >2.0 0.031 >8.00.031 >8.0 Candida krusei ATCC6258 0.25 0.25 0.25 1.0 0.13 0.25 RhizopusATCC11145 0.25 0.5 8.0 >8.0 0.13 >8.0 oryzae Cryptococcus ATCC240670.016 0.063 0.016 1.0 0.016 0.25 neoformans Chaectomium ATCC44699 0.0310.13 0.5 1.0 0.13 0.25 globosum Fusarium ATCC16106 0.130.5 >8.0 >8.0 >8.0 >8.0 graminerarum Penicillium ATCC9480 0.063 0.13 1.02.0 0.063 0.13 chrysogenum Cladosporium NCPF2564 0.016 0.016 ND ND ND NDherbarum Trichophyton ATCC10218 ND 0.031 <0.008 0.063 <0.008 0.031rubrum Table footnotes: MIC₅₀/MIC₁₀₀ = concentration required for 50%and 100% inhibition of fungal growth by visual inspection (CLSI). ND:not determined

Inhibitory activities on alveolar invasion of Aspergillus fumigatus(azole sensitive strain: NCPF2010; and azole resistant strain TR34/L98H)were determined by measuring galactomannan (GM) in the bottom chamber ofhuman alveoli bilayers 1 day post infection. Administration of Compound(Ia) to the apical chamber produced concentration-dependent inhibitionof GM levels in the bottom chamber, with maximum effects exceeding 90%for both strains (Tables 6 and 7).

TABLE 6 Effects of Compound (Ia) and Posaconazole on the invasion ofAspergillus fumigatus (azole susceptible strain: NCPF2010) into thelower chamber in human alveoli bilayers (transwells). MIC ValuesTreatment for treatment indicated (nM) (Test Compound) MIC₅₀ MIC₉₀Posaconazole 155 212 Compound (Ia) 154 185 Table Footnote: n = 3.

TABLE 7 Effects of Compound (Ia) and Posaconazole on the invasion ofAspergillus fumigatus (azole resistant strain: TR34/L98H) into the lowerchamber in human alveoli bilayers (transwells). MIC Values Treatment fortreatment indicated (nM) Test Compound MIC₅₀ MIC₉₀ Posaconazole 315 793Compound (Ia) 261 417 Table Footnote: n = 1

When the inhibitory activities were monitored for several days postinfection, the early inhibitory effects of monotherapy with eitherCompound (Ia) (0.1 μg/mL in the upper chamber) or posaconazole (0.01μg/mL in the lower chamber) were found to disappear rapidly (Table 8).In contrast, the combined treatment with Compound (Ia) in the upperchamber and posaconazole in the lower chamber (as above) led tosustained inhibition of invasion post infection. Consequently, the DFB₅₀for the combination treatment was 3.63 days, much longer than the valuesfor either compound alone (Table 8). This synergistic or at leastadditive effect of combination therapy also occurred when treatment withCompound (Ia) was combined with that of itraconazole, voriconazole orcaspofungin (results not shown).

TABLE 8 Effects of Compound (Ia), Posaconazole and the treatmentcombination on Aspergillus fumigatus (NCPF2010) invasion into the lowerchamber in human alveoli bilayers (transwells). GM Levels in the LowerChamber for Treatments Indicated OD value (% inhibition vs. control)Treatment Posaconazole² Compound (Ia)¹ Lower Combination Day VehicleUpper Chamber Chamber Treatment 0 0 0 0 0 1 0.73 0.24 (66) 0.074 (89)0.019 (97) 2 1.73 1.71 (1.5) 1.71 (1.5) 0.11 (94) 3 1.82 1.67 (8.6) 1.70(7.1) 0.18 (90) 4 1.65 1.68 (−1.6) 1.70 (−3.0) 1.34 (19) 5 1.64 1.63(0.2) 1.69 (−3.6) 1.72 (−5.3) 6 1.55 1.57 (−1.8) 1.59 (−3.0) 1.62 (−4.6)7 1.66 1.54 (7.1) 1.62 (2.5)  1.59 (4.4) DFB₅₀ Values for 1.04 1.16 3.63treatments indicated Table footnotes: ¹Dosed at 0.1 μg/mL; ²Dosed at0.01 μg/mL; DFB₅₀: Days taken to reach a fungal burden of 50% of control

In addition, this combination treatment has been tested in bilayersinfected with the azole resistant strain of Aspergillus fumigatus:TR34-L98H (Table 9). Monotherapy with Compound (Ia) (0.3 μg/mL) in theupper chamber or with posaconazole (0.1 μg/mL) in the lower chambershowed limited benefit. In contrast, the combination of Compound (Ia)and posaconazole showed marked inhibitory effects on fungal invasioninto the lower chamber.

TABLE 9 Effects of Compound (Ia), Posaconazole and the treatmentcombination on Aspergillus fumigatus (azole resistant strain: TR34-L98H)invasion into the lower chamber in the alveolar bilayer cell system(transwells). GM Levels in the Lower Chamber for Treatments Indicated ODvalue (% inhibition vs. vehicle control) Treatment Compound (I)¹Posaconazole² Combination Day Vehicle Upper Chamber Lower ChamberTreatment 0 0 0 0 0 1 0.63 0.016 (98) 0.016 (98) 0.014 (98) 2 1.11  0.84(24) 0.73 (35) 0.015 (99) 3 1.06   1.01 (4.6) 1.16 (−10) 0.020 (98)DFB₅₀ Values for 1.53 1.94 >3 treatments indicated Table footnotes:¹Dosed at 0.3 μg/mL; ²Dosed at 0.1 μg/mL; DFB₅₀: Days taken to reach afungal burden of 50% of control

When given intranasally to immunocompromised, neutropenic mice, on days1, 2 and 3 following inoculation (therapeutic treatment), Compound (Ia)showed some protection against body weight loss, measured over 3 days,caused by infection with Aspergillus fumigatus at lower doses than wererequired of posaconazole (Table 10).

TABLE 10 The Effects of Treatment with Compound (Ia) or Posaconazole onthe body weight loss of immunocompromised, neutropenic mice, caused byAspergillus fumigatus infection. Body weight loss^(1,2) Treatment DrugConc (% Inhibition of weight loss) Regimen² (mg/mL) Day 2 Day 3 Vehiclenone 5.5 ± 1.5 10.7 ± 1.8 plus Spores Compound (Ia) 0.0032 4.2 ± 1.2(24) 9.7 ± 2.1 (9) 0.016 3.6 ± 1.2 (35) 9.7 ± 3.2 (9) 0.08 2.8 ± 2.4(49) 8.3 ± 6.7 (22) Vehicle none 4.7 ± 2.1  9.3 ± 2.7 plus SporesPosaconazole 0.4 5.4 ± 0.4 (−15) 9.2 ± 4.0 (1) 2 3.9 ± 1.3 (17) 7.1 ±1.9 (24) 10 3.9 ± 1.3 (17) 4.3 ± 1.8 (54) Table Footnotes: ¹% weightloss caused by infection with Aspergillus fumigatus compared with animalweight on day 1 when treatment was started; ²Two separate studiesconducted.

Furthermore, therapeutic treatment with Compound (Ia), showed superioreffects to posaconazole on fungal load in the lung, on galactomannanconcentrations in serum and Aspergillus fumigatus DNA content in lungs.These data for Compound (I) are shown in Table 11 and FIGS. 1, 2 and 3.

TABLE 11 The Effects of Prophylactic and Therapeutic Treatment withCompound (Ia) on CFU in lung, on galactomannan concentrations in serumand on Aspergillus DNA in the lungs of Aspergillus fumigatus infected,immuno-compromised, neutropenic mice. Treat- Drug % Inhibition ofresponse ment Conc DNA in Lung Regi- (μg/ CFU GM in serum (pg/50 ng menmL) (/mg of lung) (COI) mouse DNA) Vehicle None 31.7 ± 17.4 3.38 ± 2.0270.7 ± 41.3 plus Spores Com- 3.2 25.8 ± 20.8 (19) 2.85 ± 2.76 (16) 41.7± 29.0 (41) pound 16 24.2 ± 15.8 (24) 3.01 ± 2.14 (11) 56.1 ± 53.4 (21)(Ia) 80 9.30 ± 5.20 (71) 0.53 ± 0.38 (84) 4.10 ± 4.60 (94) TableFootnotes: The data for fungal load are shown as the mean ± standarderror of the mean (SEM; n = 6).

The ID₅₀ values for posaconazole and Compound (Ia), administeredtherapeutically in independent experiments, are also presented below(Table 12).

TABLE 12 ID₅₀ values for Therapeutic Treatment with Posaconazole andCompound (Ia) on fungal load in the lung on galactomannan concentrationsin serum and on Aspergillus fumigatus DNA content in lung tissue, inAspergillus fumigatus infected, immuno-compromised, neutropenic mice.ID₅₀ Values for response indicated Drug substance (mg/mL) (TherapeuticLung Fungal GM in A. fumigatus Regimen) Load serum DNA in Lung Compound(Ia) 0.051 0.047 0.050 Posaconazole 0.39 0.59 Nt Table Footnotes: nt:not tested

Therapeutic treatment with Compound (Ia) was also found to inhibit serumcytokine concentrations in Aspergillus fumigatus infected,immunocompromised, neutropenic mice. (Tables 13 and 14; FIGS. 1, 2 and3). The calculated ID₅₀ values for inhibition of serum cytokine levels(Table 14) are very similar to those observed for lung fungal load,serum galactomannan concentrations and for lung Aspergillus fumigatusDNA content (above).

TABLE 13 The Effects of Therapeutic Treatment with Compound (Ia) on IL-6and TNFα levels in the serum of Aspergillus fumigatus infected,immunocompromised, neutropenic mice. Conc of Biomarkers (pg/mL)Treatment Drug Conc (% Inhibition) Regimen (μg/mL) IL-6 TNFα Vehiclenone 298 ± 142 35.3 ± 10.1 plus Spores Compound (Ia) 3.2  247 ± 185 (17)28.1 ± 13.8 (20) 16  262 ± 185 (12) 21.8 ± 14.6 (38) 80 66.5 ± 32.9 (78) 4.7 ± 1.0 (87) Table Footnotes: The data for biomarker concentrationsare shown as the mean ± standard error of the mean (SEM), N = 6.

TABLE 14 ID₅₀ values for Therapeutic Treatment with Compound (Ia) onIL-6 and TNFα levels in serum in Aspergillus fumigatus infected,immuno-compromised, neutropenic mice. Drug substance ID₅₀ Values forbiomarkers indicated (Prophylactic (mg/mL) Regimen) IL-6 TNFα Compound(Ia) 0.050 0.027

The effects of extended prophylactic dosing with Compound (Ia) inAspergillus fumigatus infected, immuno-compromised, neutropenic micewere also evaluated. Extended prophylaxis with Compound (Ia) was foundto inhibit fungal load in the lung, as well as GM concentrations in bothBALF and serum, at 25 fold lower doses than those used in a previousstudy (Table 15). Furthermore, the data suggest an accumulation ofanti-fungal effects in the lung on repeat dosing since seven days ofprophylaxis produced greater anti-fungal effects than did prophylactictreatment for a single day. The compound's persistence of action in thelung is suggested by the finding that treatment on days −7 to day 0generated superior anti-fungal effects on day 3 than those resultingfrom treatment on days −1 and 0, only.

TABLE 15 Effects of extended prophylactic dosing of Compound (Ia) onfungal load (CFU) in lung and on GM concentrations in BALF and serum inAspergillus fumigatus infected, immuno-compromised, neutropenic mice.Dose of CFU values, COI values for GM in BALF and serum TreatmentCompound and % Inhibition of responses² Regimen¹ (Ia) CFU GM in BALF GMin Serum (Days dosed) (μg/mL) (/mg of lung) (COI) (COI) Vehicle plusNone 9.2 ± 4.9 4.1 ± 0.7 3.9 ± 0.7 Spores −7 to +3 0.64 2.0 ± 3.6 (78)3.1 ± 0.85 (24) 2.6 ± 0.82 (33) −1 to +3 0.64 4.0 ± 5.2 (57) 3.9 ± 0.59(5) 3.6 ± 0.52 (8) −7 to +3 3.2 0.04 ± 0.08 (99.6) 1.5 ± 0.59 (63) 1.5 ±0.85 (62) −1 to +3 3.2 1.0 ± 1.4 (89) 3.4 ± 0.46 (17) 2.8 ± 0.24 (28) −7to 0 3.2 0.9 ± 1.1 (90) 2.9 ± 0.97 (29) 2.6 ± 0.48 (33) −1, 0 3.2 20.4 ±15.7 (−222) 4.5 ± 0.63 (−10) 4.7 ± 0.65 (−21) Table footnotes: ¹The Nvalue was five for all vehicle and drug treated groups; ²The data forfungal load and GM levels are shown as the mean ± standard error of themean and the percentage inhibition, with respect to vehicle.

In Vivo Pharmacokinetics

It is a commonly used procedure for pulmonary, therapeutic agents to bedosed into the lungs of animals, for example mice, and plasma collectedat various time points after dosing in order to characterise theresulting systemic exposure to the administered compound.

The compound of the invention may be tested in such above mentioned invivo systems.

Summary of the Biological Profile of Compound (I)

Compound (I) in the form of all four stereoisomers has been found to bea potent inhibitor of Aspergillus fumigatus planktonic growth andbronchial epithelial cell infection. Compound (Ia) inhibited the growthof posaconazole-resistant and voriconazole-resistant Aspergillusfumigatus isolates, demonstrating greater potency than posaconazole,voriconazole and Amphotericin B against these strains. A wide range ofother pathogenic fungi were also found to be sensitive to Compound (Ia).Synergistic or at least additive effects have been shown for Compound(Ia) in combination with posaconazole, itraconazole, voriconazole andcaspofungin. In vivo, in Aspergillus fumigatus infected,immunocompromised, neutropenic mice, Compound (Ia), demonstrated potentinhibition of Aspergillus fumigatus infection, as well as the associatedlung immune response when dosed therapeutically or prophylactically.Compound (Ia) was also efficacious in reducing infection-dependent bodyweight loss. These inhibitory effects were superior to those ofposaconazole. It is clinically significant that the beneficialanti-fungal effects of Compound (I) are observed in a therapeuticsetting.

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The invention claimed is:
 1. A process for preparing a compound offormula (I):

or a pharmaceutically acceptable salt thereof wherein the processcomprises reacting a compound of formula (6):

wherein R is H; with 2-aminocyclohexanol or a salt thereof.
 2. Theprocess according to claim 1 wherein the compound of formula (I) isprepared in the form of a singular stereoisomer selected from compounds(Ia) and (Ib):

or a pharmaceutically acceptable salt of any one thereof.
 3. The processaccording to claim 2 wherein the compound of formula (I) is compound(Ia):

or a pharmaceutically acceptable salt thereof.